Project Summary In this proposal we will apply biochemical and single-molecule approaches to understand the mechanism of non-homologous end joining (NHEJ), the primary DNA double strand break (DSB) repair pathway in human cells. During NHEJ, core factors, end processing factors and other accessory factors tether DNA ends together and ultimately ligate them. While the biochemical activities of these individual factors are known to varying extents, it remains poorly understood how these factors assemble into a synaptic complex and how their various enzymatic activities are coordinated. To that end, we will apply single-molecule imaging approaches in Xenopus egg extract to directly follow NHEJ complex formation and end synapsis in real time during a physiological repair reaction. Completion of the specific aims below will provide an increased mechanistic understanding of NHEJ, which will aid efforts to therapeutically target NHEJ and to modulate repair outcomes during CRISPR-Cas gene editing. Aim 1: How does the synaptic complex assemble and evolve during NHEJ? Upon DSB formation it is critical that DNA ends are rapidly synapsed so as to prevent the ends from diffusing apart and joining with the wrong partner. We have shown that paired ends pass through two distinct synaptic states during repair. Initially ends are held in a relatively unstable long-range synaptic complex before transitioning to a stable short-range synaptic complex in which the ends are poised to be ligated. In this aim we will determine the unique sets of intermolecular interactions that characterize the synaptic complexes and describe how these interactions evolve during repair. In particular, we will elucidate how the core NHEJ factors XLF, XRCC4 and LIG4 contribute to end synapsis and determine how accessory factors facilitate assembly of the synaptic complexes. Aim 2: How do end processing factors gain access to DNA ends? To minimize aberrant end processing and resection, DNA ends are rapidly bound by Ku and other factors. In the prior funding period, we showed that even NHEJ-associated end processing is restricted until formation of the ligation-competent short-range synaptic complex. This regulation prioritizes ligation over error-prone end processing. In this aim we will elucidate the molecular steps that enable end deprotection and allow for end processing. Furthermore, we will determine how Ku is remodeled on DNA ends during repair and examine the consequences on NHEJ by blocking this remodeling. Next, we will determine how different processing factors compete for DNA ends after they become accessible. Finally, we will apply our mechanistic insight into the regulation of end processing to decrease the fidelity of repair of CRISPR-Cas9 induced breaks in cells.